High Performance Liquid Chromatographic Fractionation System

⁠⁠⁠The use of a High Performance Liquid Chromatographic (HPLC) fractionation instrument is critical to NMI and has been used to maintain the certification of multiple Certified Reference Materials produced by NMI in the last decade. It is also currently being used for the provision of reference values for new ventures in the Health Sector and is an essential clean up technique for highly selective and sensitive reference measurements. The system currently in use has been assembled from a number of components many of which are well in excess of ten years old and are becoming less reliable.

The HPLC fractionation system will be used to clean-up samples for subsequent analysis of selected fractions by LC/MS and/or GC/MS. The ability to perform comprehensive 2D separations is not required; however the system will be capable of performing one or more heart cuts from the first HPLC column to a second column of different chromatographic selectivity. A typical application would involve chromatographic separation and collection of selected steroids in an extract from 10 mL of urine pre-concentrated to 55 μL. Fractions heart-cut from the first dimension must be able to be diluted with a weaker solvent prior to entering the second column to allow re-focussing. One single complete system is required.

Under complete programmed software control, the system must be able to:
* Perform automated HPLC separations on aliquots from at least 25 separate vials containing between 0.05 and 1.5 mL of sample solution. Injection volumes of 5 - 50 μL are required without loss of sample and leaving no more than 5 μL of residual solution in the vial.
* Simultaneously produce independent binary solvent gradients for two separate chromatographic pathways at flow rates of between 0.1 and 5 mL per minute.
* Maintain the temperature of two HPLC columns independently at between ambient temperature and at least 50 °C. The ability to operate to higher temperatures is desirable.
* Directly transfer and efficiently trap selected portions of the effluent from the first chromatographic column onto the head of a second column to permit subsequent separation of trapped components in a second chromatographic dimension.
* Monitor the UV/visible absorbance of the effluent of either one of the chromatographic columns as required at two or more selected wavelengths simultaneously during the course of each sample separation.
* Collect fractions eluted from either of the chromatographic columns based on retention time. At least 96 fractions of up to 5 mL must be collectable in a given automated sample sequence. The ability to collect fractions in 100 mm x 13 mm i.d. tubes is desirable for compatibility with existing workflows.
* System control software should have a facility for graphically displaying the timing of solvent path changes as well as solvent gradients and fraction collection windows.